1. Field of the Invention
This invention relates to an immunobiosensor for sensing antigens, a process for producing such an immunobiosensor, and a process for sensing antigens with said immunobiosensor. Immuno-biosensing techniques to measure specific antigen-antibody binding reactions are important in the development of biosensor applications in biotechnology, diagnosis, medicine and food technology.
2. Description of Prior Art
Immunobiosensors are of great interest due to their potential utility as specific and direct detection tools and their simplicity compared to standard immulogic tests, including enzyme linked immunosorbent assays and immunoradiometric assays. See SIBBALD, A. J., J. Mole. Elec. 1986, 2, 51-83. Immunobiosensors, like other types of biosensors, utilize a molecular recognition element comprising a transduction system coupled to a receptor. (Buch, R. M. et al., Analytic Chemistry, 1989, 61, 533A; Thompson, M. et al., Analytic Chemistry, 1991, 63, 393A). The molecular recognition is achieved by sensing a specific antigen-antibody binding reaction at the receptor. The transduction system identifies and responds to the changes in an optical, spectroscopic, chemical, electrochemical, radiochemical, or electrical parameter of the receptor environment due to the specific binding (Sutherland, R. M. et al., Clinical Chemistry, Winston-Salem, N.C., 1984, 30, 1533-1538; and Aizawa et al., Proc. Int. Meet. Chem. Sens. 2nd, 1986, 6-30, 622-625).
Transduction systems that use capacitive changes for sensing antigen-antibody binding reactions are relatively new. See for example, Bataillard, P. et al., Analytical Chemistry, 1988, 60, 2374-2379. Localized capacitances can change with local solution conductivity or as a result of the change in the dielectric constant caused by the antigen-antibody complex formation. Newman et al., W.D. Proc. Int. Meet. Chem. Sens., 2nd, 1986, 6-23, 5966-598 teaches the use of capacitance measurements with photolithographically defined electrodes to develop an immunobiosensor. Bataillard et al. teaches the use of capacitances measurements for the direct detection of antigen-antibody binding. Measurements of a.c. impedances, which includes capacitive changes, however, are not widely employed in immunobiosensors technology (Hesketh, P. J. et al., Sensors and Actuators B, 1993, 13-14, 749-751). Similarly, measurements of d.c. impedances are not widely employed in biosensor signal transduction.
The molecular recognition receptor of an immunobiosensor is prepared by immobilizing the biological recognition element, typically antibodies, onto a substrate material. This is a critical step because the antibody activity must remain high after immobilization and binding of antigen to antibody should occur in a manner that reduces interferences (Turner, A.P.F., Biosensor: Fundamentals and Applications, Oxford University, 1987, 1-359). U.S. Pat. No. 5,077,210 teaches a method for covalently immobilizing active agents such as proteins on suitable substrates. In accordance with one embodiment, the enzyme acetylcholine esterase is immobilized on a small strip of platinum (Pt) foil. The activity of the immobilized enzyme is measured amperometrically or potentiometricly with the Pt strip as an electrode.
U.S. Pat. No. 5,269,903 teaches a micro-electrode in which enzyme molecules or biologically active substances are immobilized. The micro-bioelectrode is formed by depositing fine particles of a conductive material, such as platinum black, and a biologically active substance on the surface of an electroconductive material, such as Pt. The biologically active substance is then immobilized with a crosslinking agent so as to form an insoluble crosslinked substance in a porous deposition formed by the conductive material.
U.S. Pat. No. 5,074,977 teaches a measuring instrument having a reversibly selective binding protein immobilized upon an insulated-gate region of a field-effect transistor located on a sensor. The '977 patent further discloses immobilizing a binding protein on a silicon dioxide surface of a semi-conducting element. The '977 patent also teaches that the binding protein may also be immobilized by metal such as aluminum, antimony, chrome, gold, platinum or silver to construct an electrode which measures conductance, resistance or potentiometric changes in response to the operation of the binding protein.
U.S. Pat. No. 4,822,566 teaches an electrochemical sensor for determining the concentration of an analyte in a fluid medium. The sensor has two (2) conductors which are coated with a thin electrically insulating passivation layer. Molecules of a binding agent are immobilized on the passivation layer with linking molecules, thereby forming an "open" capacitor. A fluid to be tested for a particular analyte is introduced onto the "open" capacitor. The presence of analyte molecules within the fluid causes a change in the capacitance of the sensor allowing the total concentration of analyte in the test fluid to be determined.
U.S. Pat. No. 5,053,225 teaches a water-insoluble functional organic thin film having a binder with an organic compound and a lipid. The organic thin film is stable and maintains high sensing operability when used with an organic thin film sensor, such as a biosensor.
U.S. Pat. No. 5,242,828 teaches a biosensing surface with a metal film of a free electron metal such as copper, silver, aluminum or gold. A monolayer of an organic molecule X-R-Y is applied to the metal surface. The monolayer binds a desired biospecific molecule or molecular structure which interacts selectively with one or more biomolecules. The molecules may be crosslinked to the monolayer.
U.S. Pat. No. 5,137,827 teaches a method for detecting the occurrence of a binding or complex-forming reaction between specific substances by utilizing the binding reaction to modify an electrical circuit, and then measuring a change in the electrical state of the circuit. A diagnostic element having a layer of antigens coated onto a non-conductive base between a pair of electrical conductors is also disclosed. Antibodies to be tested are bound to fine electrically conductive metallic particles and introduced onto the layer of antigens. Antibodies which react with the layer of antigens bind the electrically conductive particles to the antigen layer and modify the circuit formed by the diagnostic element.
U.S. Pat. No. 5,171,779 teaches a method of immobilizing proteins on a polymeric matrix by plasma activation. The immobilized protein is used in biotransformation or biosensing instruments, as well as for immunoassays.
Finally, U.S. Pat. No. 5,200,051 teaches a method for the microfabrication of electronic devices which have been adapted for the analyses of biologically significant analyte species. Also disclosed is a method for electrochemically detecting a particular analyte species using a substrate/label signal generating pair which produces a change in the concentration of certain electro-active species.
Kasapbasiolgu, B., M.S. Thesis, University of Illinois, Chicago, 1992, 1-63 teaches the immobilization of anti-Staphylococcus Enterotoxin B (SEB) antibodies onto an ultra-thin Pt film through physical adsorption. An impedance decrease was observed during the binding of Staphylococcus Enterotoxin B (SEB) to anti-SEB in phosphate buffered saline (PBS). However, instability of the anti-SEB layer resulted in drift and non-selectivity in the sensor signal. In addition, physically absorbed antibody molecules are likely to leave the substrate surface during sensing. Furthermore, when the antibody is immobilized by entrapment, the antigen-antibody binding reaction is hampered due to both the diffusion barrier experienced by the antigen and escape of antibodies through surface pores (Turner, A. P. F., Biosensors; Fundamentals and Applications, Oxford University, 1987, 1-359).
Immobilization through covalent binding using crosslinker molecules is known for building stable layers of antibodies onto substrates surfaces. See, for example, Bhatia et al., Analytic Biochemistry, 1989, 178, 408-413. Bataillard, P. et al., Analytic Chemistry, 1988, 60, 2374-2379 teaches that anti-.alpha.-fetoprotien covalently bound to a silanized glass surface using the crosslinker glutaraldehyde retained its activity. Bhatia et al., Analytic Biochemistry, 1989, 178, 408-413 teaches preparation of a receptor for goat-IgG by covalently binding anti-goat-IgG to a glass surface using N-.gamma.-maleimidobutyloxy succinimide ester, GMBS as the crosslinker.